Each year 500,000 infants are born prematurely in the U.S. Premature infants have a 3-times higher risk for developing an autism spectrum disorder (ASD), and the prevalence of ASDs approaches 25% in the very most prematurely-born infants. Premature infants have been shown to experience chronic hypoxia during a period of development when axon connections are forming; and the extent of hypoxic exposure for premature infants correlates with the risk for ASDs. Ex-premature infants who develop ASDs lack conspicuous brain abnormalities, but have evidence of decreased brain serotonin (5-HT) and alterations in connectivity. Our work addresses the gap in understanding the mechanism by which hypoxia disrupts axon connections. We investigate a novel role for 5- HT: sensing a developmental perturbation (hypoxia), and in response altering neural circuitry. Work from our lab has shown that hypoxia disrupts axon pathfinding in vertebrates (Stevenson et al. 2012). Hypoxia can disrupt 5-HT signaling, and recent in vitro evidence demonstrates that 5-HT can modulate axon guidance. Our hypothesis is that developmental hypoxia disrupts axon pathfinding acting through serotonin. We have developed a powerful platform to test our hypothesis. We use studies in zebrafish, combining the relevancy of vertebrate CNS structures and genes, with rapidity and efficiency for testing molecular mechanisms. We have generated unique and novel fluorescent reporter and expression/misexpression lines; and capability to generate large numbers of animals for sufficient statistical power. Our experiments combine knock- down, knock-out, and misexpression of genes; pharmacological manipulations; and an established hypoxia model. Our preliminary data shows that 5-HT2 receptors (htr2) are expressed in commissural foxP2 neurons, and that pharmacological blockade of htr2 receptors causes midline axon pathfinding errors. We use knock-down of htr2 in the foxP2 neurons to confirm the role of 5-HT on axon guidance in vivo (Aim 1). We genetically ablate the 5-HT source neurons (raphe nucleus) to demonstrate that the pathfinding is regulated by 5-HT; and we test whether 5-HT's effect is mediated by the guidance receptor ephrinB2a which is expressed in foxP2 neurons. In Aim 2 we will study whether 5-HT is a sensor for developmental hypoxia to regulate circuit and behavior development. 5-HT circuits are known to be involved in anxiety-related behaviors dysregulated in ASDs, and our preliminary work has found that hypoxia causes a persistent decrease in 5-HT expression. We will characterize 5- HT's role in hypoxia axon pathfinding errors by rescuing pathfinding using the 5-HT reuptake inhibitor fluoxetine. To determine if 5-HT circuitry changes modulate behavior, we will compare anxiety behavior (thigmotaxis/wall- hugging) in control or experimental animals exposed to hypoxia or to 5-HT pharmacological blockade; to hypoxia animals treated with fluoxetine; or to animals with 2-photon ablation of foxP2 axons. Our approach provides important mechanistic insights into the effects of hypoxia accompanying prematurity, and its involvement in disruptions of CNS connectivity associated with autism spectrum disorders.